Broad spectrum preservation blends

ABSTRACT

A composition having effective broad spectrum preservation activity comprising benzyl alcohol, salicylic acid, sorbic acid and a compound selected from the group consisting of 1,3-propanediol, glycerin and combinations thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/234,456, filed on Aug. 17, 2009, the disclosureof which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to broad spectrum preservation blends. Inparticular, the present invention relates to preservation blends thatincorporate benzyl alcohol, salicylic acid, sorbic acid and an additive.The additive is selected from the group consisting of 1,3-propanol,dehydroacetic acid, glycerin and a combination thereof. The preservationblends of the invention are stable at low temperatures and have a highefficacy against a broad spectrum of microorganisms at a wider thanexpected pH range.

Brief Description of Art

Preservatives have wide applications in fields like personal care,industrial, health and hygiene, pharmaceutical and wood protection.Preservatives can be a single agent or a blend of multiple agents.

Ideally, a preservative has broad-spectrum activity against all types ofmicroorganisms at various pH levels. The preservative should also havehigh efficacy so that a minimum amount of the preservative can be usedto save cost and to avoid or reduce any possible adverse effects causedby the preservative. Also, it is desirable that the preservative isstable to any changes in temperature encountered during manufacturing,packaging, and shipping as well as during storage of the preservative.Further, an ideal preservative is physically and chemically compatiblewith ingredients of different application systems so that onepreservative can suitably be incorporated in various products.

Benzyl alcohol, salicylic acid and sorbic acid are known preservativeagents, but individually they are of limited usefulness with regards tobroad spectrum activity because these actives are known to have pooractivity at more neutral and alkaline pH. In addition, salicylic acidand sorbic acid can be difficult to solubilize at high concentrations,thus making an effective concentrate difficult to achieve.

Certain preservative blends containing either one or more of benzylalcohol, salicylic acid, or sorbic acid are known. For example,Optiphen® BSB-N from ISP is a combination of benzoic acid, sorbic acidand benzyl alcohol and glycerin. Sharomix® 705 from Sharon Laboratoriesis a liquid blend of benzyl alcohol, benzoic acid, sorbic acid anddehydroacetic acid. However, the application of these preservatives islimited because they are only suitable for low pH systems (up to pH6.5).

US patent application publication 2009/0123577 to Air Liquide Sante, thedisclosure of which is incorporated herein in its entirety, discloses aliquid concentrate for preserving cosmetics including a carboxylic acidcomponent (A) containing at least two carboxylic acids selected frombenzoic acid, propionic acid, salicylic acid, sorbic acid,4-hydroxybenzoic acid, dehydroacetic acid, formic acid and10-undecylenic acid; an alcohol component (B) selected fromphenoxyethanol, benzyl alcohol. Unfortunately, the preservativeconcentrate disclosed in the publication uses up to 40% of water assolvent. As water freezes at 0° C., this may cause difficulties inhandling the disclosed concentrate at low temperatures. Further, thedisclosed preservatives are suitable for use in systems having a pH ofless than 7, in particular less than 6.

Accordingly, there is a continuing need for another preservative, whichis stable at low temperatures, and which has a high efficacy against abroad spectrum of microorganisms at a wide pH range. The presentinvention provides one answer to that need.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a composition havingeffective broad spectrum preservation activity comprising (a) benzylalcohol, (b) salicylic acid, (c) sorbic acid and (d) a compound selectedfrom the group consisting of 1,3-propanediol, glycerin and a combinationthereof. In the composition, component (a) is present at a concentrationof from about 70% to about 90% by weight, component (b) is present at aconcentration of from about 1% to 15% by weight, component (c) ispresent at a concentration of from about 1% to 4% by weight, andcomponent (d) is present at a concentration of from about 1% to 15% byweight, provided that the total amount of components (b) and (c) is nomore than 15% by weight, all based on the total weight of thecomposition. The composition may optionally contain dehydroacetic acid.

Still another aspect of the present invention is directed to acomposition having effective broad spectrum preservation activitycomprising benzyl alcohol, salicylic acid, sorbic acid, and glycerinwherein benzyl alcohol is present at a concentration of from about 77%to about 86%, salicylic acid is present at a concentration of from about8% to about 11%, sorbic acid is present at a concentration of from about2.5% to about 3.5% and glycerin is present at a concentration of fromabout 3% to about 5% by weight, based on the total weight of thecomposition.

Yet another aspect of the present invention is directed to topicalformulations containing the preservative of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a stable preservation blend compositionhaving effective broad spectrum preservation activity at neutral, acidicand alkaline pH. The composition contains: (a) benzyl alcohol, (b)salicylic acid, (c) sorbic acid, and (d) a compound selected from thegroup consisting of 1,3-propanediol, glycerin and a combination thereof.

The amount of components (a)-(d) of the preservation blend according tothis invention may vary. Preferably, benzyl alcohol constitutes fromabout 70% to about 90% by weight, more preferably, about 77% to about86% by weight, based on the total weight of the composition. Salicylicacid is present at a concentration of from about 1% to about 15%,preferably from about 3% to 12%, more preferably from 8% to 11%; sorbicacid is present at a concentration of from about 1% to about 4%,preferably from 2.5% to about 3.5% by weight, and component (d) ispresent at a concentration of from 1% to 15%, preferably from 1% to 10%by weight, more preferably from 2% to 5%, based on the total weight ofthe composition. Preferably, the total amount of components (b) and (c)is no more than 15% by weight based on the total weight of thecomposition. In some embodiments, the total weight percentages ofcomponents (a), (b), (c) and (d) combined is 100%.

Optionally, the preservation blend of the invention may additionallycontain dehydroacetic acid. If present, the total amount of salicylicacid, sorbic acid, and dehydroacetic acid is no more than 15 wt % basedon the total weight of the preservation blend. In one embodiment, theweight ratio of dehydroacetic acid to component (d) of the preservationblend is from about 3:1 to about 1:1.

In some embodiments of the invention, the preservative blends are freeor substantially free of water. In other embodiments, the blends arefree or substantially free of benzoic acid. As used herein, “essentiallyfree” is intended to mean that the composition preferably contains lessthan 1000 ppms, more preferably less than 100 ppms, and most preferablyzero ppms, of water or benzoic acid.

In one preferred embodiment, the invention provides a preservation blendcomposition comprising from about 77 wt % to about 86 wt % of benzylalcohol, from about 8 wt % to about 11 wt % of salicylic acid, fromabout 2.5 wt % to about 3.5 wt % of sorbic acid and from about 2 wt % toabout 5 wt % of glycerin by weight, based on the total weight of thecomposition.

As used herein, the term “effective preservation activity” means thatits activity is such that the composition or formulation is protectedfor a sustained period of time, in particular during the so-called“shelf life” of the product. The “shelf-life” of a product is determinedaccording to methods generally known in the art.

The term “broad spectrum” as used in this specification and claims meansa preservative having good preservation properties against a widespectrum of microorganisms that commonly will cause deterioration orspoilage of personal care products, such as cosmetics, and variousproducts with other applications such as pharmaceutical compositions,wood preservative systems, industrial, and health and hygiene.

The preservation blends of the invention have a relatively high organicacid level, yet are stable at low temperatures. They are effective atlow concentrations and have a broad-spectrum of activity against varioustypes of microorganisms. The preservative blends of the invention alsohave surprisingly good antimicrobial performance at neutral, acidic andlow alkaline pH, and can be incorporated into a wide range offormulations.

The composition of the present invention can be made by mixing benzylalcohol, salicylic acid, sorbic acid and component (d) in any order. Itcan be suitably incorporated into various products, for example,personal care formulations, pharmaceutical compositions, woodpreservative systems, industrial, and health and hygiene products.

In one embodiment, this invention further relates to topicalformulations containing a preservation blend as defined herein.Preferably, the preservation blend is present at a concentration of fromabout 0.5% to about 1.5%, more preferably from about 0.6% to about 1%based on the total weight of the topical formulation. Topicalcompositions comprise dermatological formulations (or topicalpharmaceutical formulations), as well as cosmetic formulations. Thetopical formulations may further contain other ingredients or additivesused in dermatological or in cosmetic formulations, including otheractive ingredients.

The formulations according to the present invention are formulated intoforms that are useful in personal care products, especially inemulsions.

The topical formulations according to the present invention mayadditionally contain further ingredients or additives such as solvents,surfactants, emulsifiers, consistency factors, conditioners, emollients,skin caring ingredients, moisturizers, thickeners, lubricants, fillers,anti-oxidants, other preservatives, active ingredients, in particulardermatologically active ingredients, fragrances and the like, as well asmixtures thereof. Active ingredients as mentioned herein comprise, forexample, anti-inflammatories, anti-bacterials, anti-fungals and the likeagents. Active ingredients suited for topical applications areparticularly preferred.

Suitable surfactants comprise: alkyl sulfates e.g. sodium laurylsulfate, ammonium lauryl sulfate; sodium cetearyl sulfate; alkylsulfoacetates e.g. sodium lauryl sulfoacetate; alkyl ether sulfates e.g.sodium laureth sulfate; sodium trideceth sulfate; sodium oleth sulfate;ammonium laureth sulfate; alkyl ether sulfosuccinates e.g. disodiumlaureth sulfosuccinate; alkyl glycosides e.g. decyl glucoside; laurylglucoside; alkyl isethionates amphoterics e.g. cocamidopropyl betaine;sodium cocoamphoacetate; sodium lauroamphoacetate; disodiumlauroamphodiacetate; disodium cocoamphodiacetate; sodiumlauroamphopripionate; disodium lauroamphodipropionate; potassium orammonium salts of the aforementioned amphoterics; capryl/capramidopropylbetaine; undecylenamidopropyl betaine; lauromidopropyl betaine; andfatty alcohol polyglycol ethers.

Suitable emulsifiers are e.g. anionics as salts of fatty acids e.g.sodium stearate or sodium palmitate, organic soaps e.g. mono-, di- ortriethanolaminoeate, sulfated or sulfonated compounds e.g. sodium laurylsulfate or sodium cetyl sulfonate, saponines, lamepones; cationics asquaternary ammonium salts; nonionics as fatty alcohols, fatty acid esterwith saturated or unsaturated fatty acids, polyoxyethylenesters orpolyoxyethylenethers of fatty acids, polymers from ethylene oxide andpropylene oxide or propylene glycol, amphotherics as phosphatides,proteins as gelatine, casein alkylamidobetaines, alkyl betaines andamphoglycinates, alkyl phosphates, alkylpolyoxyethylene phoaphates orthe corresponding acids, silicone derivatives, e.g. alkyldimethiconecoplyol.

Suitable consistency factors are e.g. fatty alcohols or their mixtureswith fatty acid esters, e.g. acetylated lanolin alcohol, aluminumstearates, carbomer, cetyl alcohol, glyceryl oleate, glyceryl stearate,glyceryl stearate (and) PEG 100 stearate, magnesium stearate, magnesiumsulfate, oleic acid, stearic acid, stearyl alcohol, myristyl myristate,isopropyl palmitate, beeswax and synthetic equivalents thereof,carbomers, and the like. Suitable conditioners are e.g. alkylamidoammonium lactate, cetrimonium chloride and distearoylethylhydroxyethylmonium methosulfate and cetearyl alcohol, cetyl dimethicone,cetyl ricinoleate, dimethicone, laureth-23, laureth-4, polydecene,retinyl palmitate, quaternized protein hydrolysates, quaternizedcellulose and starch derivatives, quaternized copolymers of acrylic ormethacrylic acid or salts, quaternized silicone derivatives.

Suitable emollients are e.g. cetearyl isononanoate, cetearyl octanoate,decyl oleate, isooctyl stearate, coco caprylate/caprate, ethylhexylhydroxystearate, ethylhexyl isononanoate, isopropyl isostearate,isopropyl myristate, oleyl oleate, hexyl laurate, paraffinum liquidum,PEG-75 lanolin, PEG-7 glyceryl cocoate, petrolatum, ozokeritecyclomethicone, dimethicone, dimethicone copolyol, dicaprylyl ether,butyrospermum parkii, buxus chinensis, canola, carnauba cera, coperniciacerifera, oenothera biennis, elaeis guineensis, prunus dulcis, squalane,zea mays, glycine soja, helianthus annuus, lanolin, hydrogenated castoroil, hydrogenated coconut oil, hydrogenated polyisobutene, sucrosecocoate, stearoxy dimethicone, lanolin alcohol, isohexadecane.

Suitable skin care ingredients are e.g. plant extracts, bisabolol,anti-inflammatory agents, urea, allantoin, panthenol and panthenolderivatives, phytantriol, vitamins A, E, C, D, ceramides of animal orplant origin, lecithins, and the like.

Suitable moisturizers are e.g. butylenes glycol, cetyl alcohol,dimethicone, dimyristyl tartrate, glucose glycereth-26, glycerin,glyceryl stearate, hydrolyzed milk protein, lactic acid, lactose andother sugars, laureth-8, lecithin, octoxyglycerin, PEG-12, PEG 135,PEG-150, PEG-20, PEG-8, pentylene glycol, hexylene glycol, phytantriol,poly quaternium-39 PPG-20 methyl glucose ether, propylene glycol, sodiumhyaluronate, sodium lactate, sodium PCA, sorbitol, succinoglycan,synthetic beeswax, tri-C14-15 alkyl citrate, starch.

Suitable thickeners are e.g. acrylates/steareth-20 methacrylatecopolymer, carbomer, carboxymethyl starch, cera alba, dimethicone/vinyldimethicone crosspolymer, propylene glycol alginate,hydroxyethylcellulose, hydroxypropyl methylcellulose, silica, silicadimethyl silylate, xanthan gum, hydrogenated butylenes/ethylene/styrenecopolymer.

Suitable lubricants are e.g. adipic acid, fumaric acid and its salts,benzoic acid and its salts, glycerine triacetate, sodium or magnesiumlauryl sulfate, magnesium stearate, solid polyethylenglycol,polyvinylpyrrolidone, boric acid, mono-laurate or mono-palmitate,myristyl alcohol, cetyl alcohol, cetylstearyl alcohol, talcum, calciumor magnesium salts of higher fatty acids, mono-, di- or triglycerides ofhigher fatty acids, polytetrafluorethylen.

Suitable antioxidants are e.g. sulfites, e.g. sodium sulfite, tocopherolor derivates thereof, ascorbic acid or derivates thereof, citric acid,propyl gallate, chitosan glycolate, cysteine, N-acetyl cysteine pluszinc sulfate, thiosulfates, e.g. sodium thiosulfate, polyphenoles andthe like.

The compositions may further contain active ingredients, e.g.anti-microbials, anti-inflammatories, plant extracts, bisabolol,panthenol, tocopherol, actives for anti-stinging, anti-irritant oranti-dandruff applications, or anti-aging agents such as retinol,melibiose and the like. Other suitable actives are e.g. Medicagoofficinalis, Actinidia chinensis, allantoin, Aloe barbadensis, Anonacherimolia, Anthemis nobilis, Arachis hypogaea, Arnica Montana, Avenasativa, beta-carotene, bisabolol, Borago officinalis, butylenes glycol,Calendula officinalis, Camellia sinensis, camphor, Candida bombicola,capryloyl glycine, Carica papaya, Centaurea cyanus, cetylpyridiniumchloride, Chamomilla recutita, Chenopodium quinoa, Chinchona succirubra,Chondrus crispus, Citrus aurantium dulcis, Citrus grandis, Citruslimonum, Cocos nucifera, Coffea Arabica, Crataegus monogina, Cucumismelo, dichlorophenyl imidazoldioxolan, Enteromorpha compressa, Equisetumarvense, ethoxydiglycol, ethyl panthenol, farnesol, ferulic acid,Fragaria chiloensis, Gentiana lutea, Ginkgo biloba, glycerin, glyceryllaurate, Glycyrrhiza glabra, Hamamelis virginiana, heliotropine,hydrogenated palm glycerides, citrates, hydrolyzed castor oil,hydrolyzed wheat protein, Hypericum perforatum, Iris florentina,Juniperus communis, Lactis proteinum, lactose, Lawsonia inermis,linalool, Linum usitatissimum, lysine, magnesium aspartate, Magniferaindica, Malva sylvestris, mannitol, mel Melaleuca alternifolia, Menthapiperita, menthol, menthyl lactate, Mimosa tenuiflora, Nymphaea alba,olaflur, Oryza sativa, panthenol, paraffinum liquidum, PEG-20M, PEG-26jojoba acid, PEG-26 jojoba alcohol, PEG-35 castor oil, PEG-40hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-8caprylic/capric acid, Persea gratissima, petrolatum, potassiumaspartate, potassium sorbate, propylene glycol, Prunus amygdalus dulcis,Prunus armeniaca, Prunus persica, retinyl palmitate, Ricinus communis,Rosa canina, Rosmarinus officinalis, Rubus idaeus, salicylic acid,Sambucus nigra, sarcosine, Serenoa serrulata, Simmondsia chinensis,sodium carboxymethyl betaglucan, sodium cocoyl amino acids, sodiumhyaluronate, sodium palmitoyl praline, stearoxytrimethylsilane, stearylalcohol, sulfurized TEA-ricinoleate, talc, Thymus vulgaris, Tiliacordata, tocopherol, tocopheryl acetate, trideceth-9, triticum vulgare,tyrosine, undecylenoyl glycine, urea, Vaccinium myrtillus, valine, zincoxide, zinc sulfate.

The preservative blends of the invention can be used in emulsions (bothoil-in-water and water-in-oil), in aqueous solutions, in PIT (phaseinversion temperature) emulsions, in oily solutions, in foaming cosmeticformulations (foams), and in so-called multiple emulsions, e.g. intriple emulsions (such as water/oil/water emulsions).

The preservative blends of the invention can also be formulated ascreams, gels, liquids or lotions. They can be used in shampoos, hairconditioners, hair dyes, hair preparations, aftershave lotions, bathsoaps and detergents, fragrance preparations, sun care products, indoortanning products, body and hand preparations, personal cleansers,shaving preparations, tonics, dressings and other hair grooming aids,moisturizing preparations, skin care preparations, wipes and the like.These compositions can be also used in a variety of non-personal careproducts.

The topical formulations of the invention are prepared by adding otheringredients to a composition as defined herein, or addition to a mixtureof ingredients a composition as defined herein. Alternatively, saidformulations may also be made by mixing the ingredients individually orby group-wise mixing. Subsequently other specific ingredients, such asperfumes, may be added.

The present invention is further described in detail by means of thefollowing Examples. All parts and percentages are by weight and alltemperatures are degrees Celsius unless explicitly stated otherwise.

EXAMPLES Example 1 Stability of Preservative Concentrates

Procedure: Preservative blends ETC 1-6 and comparative blends A-G asshown in Table 1 and Table 2, were prepared by mixing the componentstogether with gentle heating. The blends were stored either in arefrigerator (2-4° C.) or a freezer (−17° C.), and then were allowed towarm up to room temperature. The characteristics of the blends at 2° C.and after freezing at −17° C. were summarized in Table 1 and Table 2.

Results: As shown in Table 1, when the total amount of salicylic acid,sorbic acid and dehydroacetic acid is less than 15 wt %, the use ofco-solvent, 1,3-propanediol and glycerin helps with stabilizing theseacids in benzyl alcohol at low temperatures. Although the co-solventdoes not prevent freezing of the blends at −17° C., it does enable theblends to readily thaw and redissolve without or with limited mixing.

As shown in Table 2, when the total amount of salicylic acid and sorbicacid was greater than 15%, although co-solvent 1,3-propanediol waspresent, the blends froze after storage at 2-4° C. After the blends werestored at −17° C., crystals were settled out and mixing was needed tore-dissolve the precipitates.

TABLE 1 Effect of co-solvents (1,3-propanediol and glycerin) on blendcharacteristics upon refrigeration and freezing Character- Ben- Dehy-1,3- istics at 2° C. zyl Sali- Sor- dro- pro- and after Blend alco-cylic bic acetic pane- Glyc- freezing at # hol acid acid acid diol erin−17° C. ECT 83% 11% 3% — 3% — Clear sol at 1 2° C., crystals settled outafter freeze/ thaw cycle ECT 83%  4% 3% 7% 3% — Clear sol at 2 2° C.,crystals settled out after freeze/ thaw cycle ECT 83%  8% 3% 3% 3% —Clear sol at 3 2° C., crystals formed by freeze/thaw cycle re-dis- solvewithout mixing ECT 83% 11% 3% — — 3% Clear sol at 4 2° C., very fewcrystals formed by freeze/thaw cycle all re- dissolve with- out mixingECT 83%  4% 3% 7% — 3% Clear sol at 5 2° C., crystals formed byfreeze/thaw cycle re-dis- solve without mixing ECT 83%  8% 3% 3% — 3%Clear sol at 6 2° C., least amount of crystals formed by freeze/ thawcycle all re-dissolve without mixing

TABLE 2 Effect of co-solvent (1,3-propanediol) on blend characteristicsupon refrigerated storage Characteristics Blend after storage inCharacteristics (Compar- Benzyl Salicylic Sorbic 1,3- refrigerator afterfreeze/thaw ative) alcohol acid acid propanediol (2-4° C.) cycle A 81.0%13.0% 6.0% — Froze in the cold, Very thick layer of went back intocrystals settled out, solution with went back into mixing solution afterabout 1 hour of mixing B 80.0% 13.0% 6.0% 1.0% Froze in the cold, Thicklayer of went back into crystals settled out, solution with went backinto mixing solution after about 40 minutes of mixing C 81.0% 12.5% 5.5%1.0% Froze in the cold, A layer of crystals went back into settled out,went solution with back into solution mixing after about 10 minutes ofmixing D 79.0% 13.0% 6.0% 2.0% Did not completely Very thick layer offreeze (slushy), crystals settled out, went back into went back intosolution with solution after about mixing 10 minutes of mixing E 80.0%12.5% 5.5% 2.0% Did not completely Thick layer of freeze (slushy),crystals settled out, went back into went back into solution withsolution after about mixing 10 minutes of mixing F 80.0% 12.0% 5.0% 3.0%Did not freeze, a few A thin layer of crystals ppt, went crystalssettled out, back into solution went back into with mixing solutionafter about 5 minutes of mixing G 79.0% 12.5% 5.5% 3.0% Froze, but veryA thin layer of rapidly melted to crystals settled out, give a solutionwith went back into a few crystals ppt, solution after about went backinto 10 minutes of solution with mixing mixing

Example 2 Microbiological Efficacy—1% Preservation Blends

CTFA Challenge Test Procedure: A challenge protocol similar to the CTFAmethod was followed to assess efficacy against a broad spectrum ofmicroorganisms. The five separate inocula were: Staphylococcus aureus(ATCC 6538), mixed Pseudomonas aeruginosa (ATCC 9027) and Burkholderiacepacia (ATCC 25416), mixed Klebsiella pneumoniae (ATCC 4352) andEnterobacter gergoviae (ATCC 33028), Candida albicans (ATCC 10231), anda mixture of molds: Aspergillus niger (ATCC 16404) and 2 Penicillium sp.isolated from cosmetic products. Samples (35 grams each) were inoculatedwith approximately 2,000,000 bacteria per gram or 100,000 yeast cells ormold spores per gram. Individual challenges were prepared from overnightslants of bacteria and yeast cultures and from heavily sporulating moldcultures, 7 to 10 days old. All samples were plated quantitatively forviable organisms after 24 hours and weekly for up to 4 weeks. Samplesinoculated with mold spores were also plated after 48 hours. Sampleswere re-challenged after four weeks (or sooner where appropriate) andthe same sampling regime followed.

Recommended “Pass” Criteria: CTFA recommends at least a 99.9% reductionof vegetative bacteria and at least a 90% reduction of yeasts and moldswithin 7 days following each challenge with no increase in countthereafter.

Test Formulations:

The formulations used to demonstrate the efficacy of our blends were asfollows.

i. Oil in water lotion, pH 6.5, AR12-034

ii. Hair Conditioner, pH 3.99, AR13-069 (same as AR5-024)

iii. Make-up Remover, pH 5.15, AR12-067 (Ref # KKL9-181)

iv. Lotion, pH 7.85, KKL 14-46

v. Water in Oil Emulsion, pH N/A, AR12-068

vi. Make-up Remover, pH 8.1, KKL 14-45

The preservative concentrates were added to these formulations to give afinal concentration of 1%.

Summary of Test Results:

(i) Oil in Water Lotion, pH 6.5, AR12-034 (Data Shown in Tables 3Through 5d)

Initial screen with ECT1, ECT2 and ECT 3 blends showed excellentreduction of all challenge organisms occurred within 24 hours in thepreserved samples and all were reduced to <10 cfu/g within one week,whereas the unpreserved controls had high counts during the test period.

TABLE 3 Inoculum-Colony Forming Units Added per Gram (CFU/g) of Oil inwater lotion pH 6.5 Challenge #1 Challenge #2 Organism CFU/g CFU/g S.aureus 2.9 × 10⁶ 1.6 × 10⁶ K. pneumoniae + E. gergoviae 4.0 × 10⁶ 2.9 ×10⁶ P. aeruginosa + B. cepacia 2.0 × 10⁶ 1.2 × 10⁶ C. albicans 8.5 × 10⁴1.6 × 10⁵ Mixed Mold 9.0 × 10⁴ 9.0 × 10⁴

TABLE 4 Inoculum Recovered from Unpreserved Oil in water lotion pH 6.5at ‘0’ Hour-Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge#2 Organism CFU/g CFU/g S. aureus 1.3 × 10⁶ 1.9 × 10⁶ K. pneumoniae + E.gergoviae 3.1 × 10⁶ 7.0 × 10⁶ P. aeruginosa + B. cepacia 7.9 × 10⁴ 4.2 ×10⁶ C. albicans 6.8 × 10⁴ 1.3 × 10⁵ Mixed Mold 8.0 × 10⁴ 7.0 × 10⁴

TABLE 5a Unpreserved Oil in water lotion pH 6.5- Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 24 48 1 2 OrganismHours Hours Week Weeks Hours Hours Week Weeks S. aureus 1.4 × 10⁶ — 4.8× 10⁵ 3.3 × 10⁴ 2.1 × 10⁶ — 1.1 × 10⁶ 6.0 × 10³ K. pneumoniae + 9.4 ×10⁵ — 3.0 × 10⁶ 1.4 × 10⁶ 5.9 × 10⁶ — 4.5 × 10⁶ 9.9 × 10⁵ E. gergoviaeP. aeruginosa + <10² — 1.6 × 10² <10 2.4 × 10⁶ — 2.8 × 10⁶ 1.8 × 10⁶ B.cepacia C. albicans 3.6 × 10⁴ — 2.0 × 10⁴ 2.6 × 10⁴ 8.0 × 10⁴ — 8.9 ×10⁴ 8.0 × 10⁴ Mixed Mold 1.8 × 10⁴ 1.1 × 10⁴ 1.7 × 10⁴ 3.0 × 10⁴ 2.8 ×10⁴ 2.0 × 10⁴ 2.0 × 10⁴ 2.8 × 10⁴

TABLE 5b Oil in water lotion pH 6.5 with 1% ECT-1- Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 24 48 1 2 OrganismHours Hours Week Weeks Hours Hours Week Weeks S. aureus <10 — <10 <10<10 — <10 <10 K. pneumoniae + <10 — <10 <10 <10* — <10 <10 E. gergoviaeP. aeruginosa + <10 — <10 <10 <10 — <10 <10 B. cepacia C. albicans 3.0 ×10¹ — <10 <10 <10 — <10 <10 Mixed Mold 1.0 × 10³ 2.0 × 10¹ <10 <10 2.0 ×10² 6.0 × 10¹ <10 <10 *Bacillus contamination

TABLE 5c Oil in water lotion pH 6.5 with 1% ECT-2 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 48 2 24 2 Organism 24 HoursHours 1 Week Weeks Hours 48 Hours 1 Week Weeks S. aureus 7.9 × 10⁴ — <10<10 4.9 × 10⁴ — <10 <10 K. pneumoniae + <10 — <10 <10 <10 — <10 <10 E.gergoviae P. aeruginosa + <10 — <10 <10 <10 — <10 <10 B. cepacia C.albicans 2.9 × 10³ — <10 <10 1.8 × 10⁴ — <10 <10 Mixed Mold 1.2 × 10⁴1.0 × 10² <10  <10* 5.0 × 10³ 8.0 × 10¹ <10 <10 *Bacillus contamination

TABLE 5d Oil in water lotion pH 6.5 with 1% ECT3- Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 48 2 24 2 Organism 24 HoursHours 1 Week Weeks Hours 48 Hours 1 Week Weeks S. aureus 1.1 × 10² — <10<10 9.0 × 10¹ — <10 <10 K. pneumoniae + <10 — <10 <10 <10 — <10 <10 E.gergoviae P. aeruginosa + <10 — <10 <10 <10 — <10 <10 B. cepacia C.albicans 1.8 × 10³ — <10 <10 1.4 × 10⁴ — <10 <10 Mixed Mold 6.0 × 10³4.0 × 10¹ <10 <10 1.7 × 10³ <10 <10 <10

(ii) Hair Conditioner, pH 3.99, AR 13-069 (Same as AR5-024) (Data Shownin Tables 6 Through 8c)

Excellent reduction of all challenge organisms occurred within 24 hoursin the preserved samples and all were reduced to <10 cfu/g within oneweek. Although S. aureus died off within one week in the unpreservedsamples, efficacy of the preservatives against this organism was obviousat the 24 hour sampling time.

TABLE 6 Inoculum-Colony Forming Units Added per Gram (CFU/g) of HairConditioner Challenge #1 Challenge #2 Organism CFU/g CFU/g S. aureus 2.1× 10⁶ 2.6 × 10⁶ K. pneumoniae + E. gergoviae 4.0 × 10⁶ 3.5 × 10⁶ P.aeruginosa + B. cepacia 3.8 × 10⁶ 4.4 × 10⁶ C. albicans 4.8 × 10⁴ 1.7 ×10⁴ Mixed Mold 1.4 × 10⁵ 1.0 × 10⁵

TABLE 7 Inoculum Recovered from Unpreserved Hair Conditioner at ‘0’Hour-Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge #2Organism CFU/g CFU/g S. aureus 9.2 × 10⁵ 1.0 × 10⁵ K. pneumoniae + E.gergoviae 1.4 × 10⁶ 2.7 × 10⁸ P. aeruginosa + B. cepacia 1.4 × 10⁶ 1.0 ×10⁸ C. albicans 3.1 × 10⁴ 1.9 × 10⁷ Mixed Mold 4.8 × 10⁴ 2.9 × 10⁴

TABLE 8a Unpreserved Hair Conditioner- Colony Forming Units per Gram(CFU/g) Challenge #1 24 48 2 3 4 Organism Hours Hours 1 Week Weeks WeeksWeeks S. aureus 3.5 × 10⁵ — <10 <10 <10 <10 K. pneumoniae + 9.4 × 10⁵ —3.4 × 10⁵ 9.0 × 10⁷ 2.2 × 10⁸ 2.6 × 10⁸ E. gergoviae P. aeruginosa + 4.9× 10⁵ — >10⁶ 2.1 × 10⁸ 6.0 × 10⁸ 3.0 × 10⁸ B. cepacia C. albicans 3.3 ×10⁵ — 3.3 × 10⁶ 2.7 × 10⁶ 1.9 × 10⁶ 2.7 × 10⁶ Mixed Mold 2.1 × 10⁴ 1.7 ×10⁴ 3.5 × 10³ 2.3 × 10³ 1.1 × 10³ 1.2 × 10³ Challenge #2 24 48 2 3 4Organism Hours Hours 1 Week Weeks Weeks Weeks S. aureus 3.5 × 10⁵ — <10<10  <10  <10  K. pneumoniae + 9.4 × 10⁵ — 1.2 × 10⁸ 5.5 × 10⁷ 1.4 × 10⁷3.5 × 10⁶ E. gergoviae P. aeruginosa + 4.9 × 10⁵ — 5.6 × 10⁸ >10⁸ >10⁸>10⁸ B. cepacia C. albicans 3.3 × 10⁵ — 2.0 × 10⁷ 2.0 × 10⁷ 6.2 × 10⁶2.8 × 10⁷ Mixed Mold 2.1 × 10⁴ 2.0 × 10⁴ 6.0 × 10³ 2.6 × 10⁴ 3.6 × 10⁴1.4 × 10⁴

TABLE 8b Hair Conditioner with 1% ECT-4 - Colony Forming Units per Gram(CFU/g) Challenge #1 Challenge #2 24 48 2 3 4 24 48 2 3 4 Organism HoursHours 1 Week Weeks Weeks Weeks Hours Hours 1 Week Weeks Weeks Weeks S.aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K. pneumoniae + <10 —<10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae P. aeruginosa + 2.0 ×10² — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia C. albicans 6.0 ×10¹ — <10 <10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold <10 <10 <10 <10<10 <10 <10 <10 <10 <10 <10 <10

TABLE 8c Hair Conditioner with 1% ECT-6 - Colony Forming Units per Gram(CFU/g) Challenge #1 Challenge #2 24 48 2 3 4 24 48 2 3 4 Organism HoursHours 1 Week Weeks Weeks Weeks Hours Hours 1 Week Weeks Weeks Weeks S.aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K. pneumoniae + <10 —<10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae P. aeruginosa + 1.2 ×10² — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia C. albicans <10 —<10 <10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold <10 <10 <10 <10 <10 <10<10 <10 <10 <10 <10 <10

(iii) Make-Up Remover, pH 5.15, AR12-067 (Data Shown in Tables 9 Through11c)

Excellent reduction of all challenge organisms occurred within 24 hoursin the preserved samples and all were reduced to <10 cfu/g within oneweek. S. aureus and the K. pneumoniae/E. gergoviae inocula died offwithin one week in the unpreserved sample. The short survival time ofthese organisms makes the results somewhat inconclusive but there issome evidence for efficacy at the 24 hour sampling time.

TABLE 9 Inoculum-Colony Forming Units Added per Gram (CFU/g) of Make-upRemover Challenge #1 Challenge #2 Organism CFU/g CFU/g S. aureus 2.1 ×10⁶ 2.6 × 10⁶ K. pneumoniae + E. gergoviae 4.0 × 10⁶ 3.5 × 10⁶ P.aeruginosa + B. cepacia 3.8 × 10⁶ 4.4 × 10⁶ C. albicans 4.8 × 10⁴ 1.7 ×10⁴ Mixed Mold 1.4 × 10⁵ 1.0 × 10⁵

TABLE 10 Inoculum Recovered from Unpreserved Make-up Remover, pH 5.15 at‘0’ Hour Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge #2Organism CFU/g CFU/g S. aureus 3.4 × 10⁴ 5.4 × 10⁵ K. pneumoniae + E.gergoviae 6.0 × 10⁵ 3.7 × 10⁵ P. aeruginosa + B. cepacia 9.4 × 10⁵ 1.8 ×10⁵ C. albicans 2.9 × 10⁵ 9.1 × 10⁵ Mixed Mold 7.0 × 10⁴ 7.3 × 10⁴

TABLE 11a Unpreserved Make-up Remover, pH 5.15 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 24 48 2 3 4 Organism Hours Hours 1 WeekWeeks Weeks Weeks S. aureus 9.0 × 10¹ — <10 <10 <10 <10 K. pneumoniae +5.3 × 10³ — <10 <10 <10 <10 E. gergoviae P. aeruginosa + 3.3 × 10⁵ — 1.8× 10⁶ 3.5 × 10⁶ 1.6 × 10⁶ 1.4 × 10⁶ B. cepacia C. albicans 1.8 × 10⁴ —1.9 × 10⁴ 8.0 × 10³ 1.7 × 10⁴ 1.2 × 10⁴ Mixed Mold 1.5 × 10⁴ 5.0 × 10⁴2.4 × 10⁴ 1.1 × 10⁴ 7.0 × 10³ 1.1 × 10⁴ Challenge #2 48 1 2 3 4 Organism24 Hours Hours Week Weeks Weeks Weeks S. aureus 7.9 × 10³ — <10 <10 <10<10 K. pneumoniae + 9.0 × 10¹ — <10 <10 <10 <10 E. gergoviae P.aeruginosa + 7.8 × 10⁶ — 2.8 × 10⁶ 6.1 × 10⁶ 5.3 × 10⁶ 7.7 × 10⁶ B.cepacia C. albicans 5.6 × 10⁴ — 6.6 × 10⁴ 3.2 × 10⁴ 2.0 × 10⁴ 1.5 × 10⁴Mixed Mold 1.0 × 10⁵ 5.0 × 10⁴ 7.7 × 10⁴ 4.2 × 10⁴ 3.3 × 10⁴ 7.0 × 10⁴

TABLE 11b Make-up Remover with 1% ECT-4, pH 5.15 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 2 3 4 24 48 1 2 3 4Organism Hours Hours 1 Week Weeks Weeks Weeks Hours Hours Week WeeksWeeks Weeks S. aureus 2.0 × 10¹ — <10 <10 <10 <10 <10 — <10 <10 <10 <10K. pneumoniae + 4.0 × 10¹ — <10 <10 <10 <10 <10 — <10 <10 <10 <10 E.gergoviae P. aeruginosa + 1.0 × 10¹ — <10 <10 <10 <10 <10 — <10 <10 <10<10 B. cepacia C. albicans <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10Mixed Mold <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10

TABLE 11c Make-up Remover with 1% ECT-6, pH 5.15 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 2 3 4 24 48 2 3 4Organism Hours Hours 1 Week Weeks Weeks Weeks Hours Hours 1 Week WeeksWeeks Weeks S. aureus 9.0 × 10¹ — <10 <10 <10 <10 <10 — <10 <10 <10 <10K. pneumoniae + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviaeP. aeruginosa + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepaciaC. albicans <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold <10<10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10

(iv) Lotion, pH 7.85, KKL 14-46 (Data Shown in Tables 12 Through 14c)

Excellent reduction of all challenge organisms occurred within 24 hoursin the preserved samples and all were reduced to <10 cfu/g within oneweek. This formula is more difficult to preserve than the other fourproducts being tested. All the challenge organisms are surviving in theunpreserved control and the P. aeruginosa/B. cepacia increased by >90%between weeks one and two.

TABLE 12 Inoculum-Colony Forming Units Added per Gram (CFU/g) of ProductChallenge #1 Challenge #2 Organism CFU/g CFU/g S. aureus 1.8 × 10⁶ 1.4 ×10⁶ K. pneumoniae + E. gergoviae 3.3 × 10⁶ 2.7 × 10⁶ P. aeruginosa + B.cepacia 4.0 × 10⁶ 2.1 × 10⁶ C. albicans 1.7 × 10⁵ 1.2 × 10⁵ Mixed Mold2.3 × 10⁵ 1.9 × 10⁵

TABLE 13 Inoculum Recovered from Unpreserved pH 7.85 Lotion at ‘0’ Hour-Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge #2 OrganismCFU/g CFU/g S. aureus 2.3 × 10⁶ 1.7 × 10⁶ K. pneumoniae + E. gergoviae2.9 × 10⁶ 3.1 × 10⁶ P. aeruginosa + B. cepacia 2.3 × 10⁶ 7.6 × 10⁷ C.albicans 1.3 × 10⁵ 1.8 × 10⁵ Mixed Mold 3.5 × 10⁵ 2.8 × 10⁵

TABLE 14a Unpreserved pH 7.85 Lotion - Colony Forming Units per Gram(CFU/g) Challenge #1 24 48 2 3 Organism Hours Hours 1 Week Weeks Weeks 4Weeks S. aureus 1.3 × 10⁶ — 1.6 × 10⁴ 1.1 × 10⁴ 2.0 × 10⁴ 3.0 × 10⁴ K.pneumoniae + 1.3 × 10⁶ — 9.5 × 10⁵ 1.8 × 10⁶ 5.4 × 10⁵ 7.0 × 10⁵ E.gergoviae P. aeruginosa + >10⁶ — 8.5 × 10⁶ 1.2 × 10⁸ >10⁸ 4.3 × 10⁷ B.cepacia C. albicans 1.1 × 10⁵ — 1.0 × 10⁵  3.0 × 10⁵* 1.9 × 10⁶ 9.0 ×10⁶ Mixed Mold 2.3 × 10⁶ 2.6 × 10⁵ 9.0 × 10⁴ 3.6 × 10⁵  5.9 × 10⁴*  1.6× 10⁴* Challenge #2 24 48 3 4 Organism Hours Hours 1 Week 2 Weeks WeeksWeeks S. aureus 1.6 × 10⁶ — 1.6 × 10⁴ 9.5 × 10³ 6.0 × 10³ 8.0 × 10³ K.pneumoniae + 4.9 × 10⁶ — 6.7 × 10⁵ 7.3 × 10⁴ 5.1 × 10³ 2.3 × 10³ E.gergoviae P. aeruginosa + 1.1 × 10⁸ — 1.6 × 10⁸ 1.2 × 10⁸ 9.5 × 10⁷ 9.8× 10⁷ B. cepacia C. albicans 2.8 × 10⁵ — 2.0 × 10⁵ 1.7 × 10⁵ 9.5 × 10⁴1.5 × 10⁵ Mixed Mold 1.1 × 10⁶ 5.2 × 10⁵* 3.7 × 10⁴ 2.8 × 10⁵ 7.0 × 10⁴7.0 × 10⁴ *Bacterial Contamination

TABLE 14b pH 7.85 Lotion with 1% ECT-4 - Colony Forming Units per Gram(CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 48 1 2 3 4 OrganismHours Hours Week Weeks Weeks Weeks 24 Hours Hours Week Weeks Weeks WeeksS. aureus 7.0 × 10¹ — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K.pneumoniae + 2.0 × 10¹ — <10 <10 <10 <10 <10 — <10 <10 <10 <10 E.gergoviae P. aeruginosa + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B.cepacia C. albicans 8.7 × 10³ — <10 <10 <10 <10 1.3 × 10⁴ — <10 <10 <10<10 Mixed Mold 1.8 × 10³ 1.9 × 10² <10 <10 <10 <10 9.0 × 10² 2.1 × 10²<10 <10 <10 <10

TABLE 14c pH 7.85 Lotion with 1% ECT-6 - Colony Forming Units per Gram(CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4 OrganismHours Hours Week Weeks Weeks Weeks Hours Hours Week Weeks Weeks Weeks S.aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K. pneumoniae + <10 —<10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae P. aeruginosa + <10 —<10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia C. albicans 2.5 × 10³ —<10 <10 <10 <10 7.7 × 10³ — <10 <10 <10 <10 Mixed Mold 6.5 × 10² 2.0 ×10¹ <10 <10 <10 <10 4.8 × 10² 5.0 × 10¹ <10 <10 <10 <10

(v) Water in Oil Emulsion, pH N/A, AR 12-068 (Data Shown in Tables 15Through 17c)

Although S. aureus and the K. pneumoniae/E. gergoviae inocula died offwithin one week in the unpreserved sample and the P. aeruginosa/B.cepacia was reduced to <10 cfu/g within two weeks, all the challengeorganisms in the preserved samples were reduced to <10 cfu/g within 24hours.

TABLE 15 Inoculum-Colony Forming Units Added per Gram (CFU/g) of ProductChallenge #1 Challenge #2 Organism CFU/g CFU/g S. aureus 1.8 × 10⁶ 1.4 ×10⁶ K. pneumoniae + E. gergoviae 2.7 × 10⁶ 2.7 × 10⁶ P. aeruginosa + B.cepacia 1.9 × 10⁶ 2.1 × 10⁶ C. albicans 1.4 × 10⁵ 1.2 × 10⁵ Mixed Mold1.5 × 10⁵ 1.9 × 10⁵

TABLE 16 Inoculum Recovered from Unpreserved Water in Oil Emulsion at‘0’ Hour - Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge#2 Organism CFU/g CFU/g S. aureus 1.8 × 10⁵ 6.6 × 10⁵ K. pneumoniae +2.5 × 10⁵ 1.2 × 10⁶ E. gergoviae P. aeruginosa + 2.9 × 10⁵ 1.8 × 10⁵ B.cepacia C. albicans 2.5 × 10⁵ 3.2 × 10⁴ Mixed Mold 2.7 × 10⁴ 9.0 × 10³

TABLE 17a Unpreserved Water in Oil Emulsion - Colony Forming Units perGram (CFU/g) Challenge #1 24 48 2 3 4 Organism Hours Hours 1 Week WeeksWeeks Weeks S. aureus 8.6 × 10⁴ — <10 <10 <10 <10 K. pneumoniae + 5.6 ×10⁴ — <10 <10 <10 <10 E. gergoviae P. aeruginosa + 3.1 × 10⁴ — 2.9 × 10³<10 <10 <10 B. cepacia C. albicans 4.6 × 10⁴ — 1.3 × 10⁴ 1.6 × 10⁴ 1.1 ×10⁴ 2.9 × 10³ Mixed Mold 1.2 × 10⁴ 2.5 × 10⁴ 9.7 × 10³ 3.3 × 10³ 4.0 ×10³ 7.0 × 10³ Challenge #2 24 48 1 2 3 4 Organism Hours Hours Week WeeksWeeks Weeks S. aureus 9.9 × 10⁴ — <10 <10 <10 <10 K. pneumoniae + 9.7 ×10⁴ — <10 <10 <10 <10 E. gergoviae P. aeruginosa + 5.6 × 10⁵ — 2.0 × 10⁴>10⁴ 6.8 × 10⁵ 3.4 × 10⁵ B. cepacia C. albicans 1.0 × 10⁵ — 5.0 × 10⁴7.2 × 10⁴ 1.4 × 10⁵ 5.3 × 10⁴ Mixed Mold 1.0 × 10⁵ 2.6 × 10⁴ 6.6 × 10⁴2.1 × 10⁴ 4.1 × 10⁴ 3.4 × 10⁵

TABLE 17b Water in Oil Emulsion with 1% ECT-4 - Colony Forming Units perGram (CFU/g) Challenge #1 Challenge #2 24 48 2 4 24 48 1 2 3 4 OrganismHours Hours 1 Week Weeks 3 Weeks Weeks Hours Hours Week Week Weeks WeeksS. aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K. pneumoniae +<10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae P. aeruginosa +<10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia C. albicans <10 —<10 <10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold <10 <10 <10 <10 <10 <10<10 <10 <10 <10 <10 <10

TABLE 17c Water in Oil Emulsion with 1% ECT-6 - Colony Forming Units perGram (CFU/g) Challenge #1 Challenge #2 24 48 2 3 4 48 2 3 4 OrganismHours Hours 1 Week Weeks Weeks Weeks 24 Hours Hours 1 Week Weeks WeeksWeeks S. aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K.pneumoniae + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae P.aeruginosa + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia C.albicans <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold <10 <10<10 <10 <10 <10 <10 <10 <10 <10 <10 <10

(vi) Make-Up Remover, pH 8.1, KKL 14-45 (Data Shown in Tables 18 Through20c)

Excellent reduction of all challenge organisms occurred within 24 hoursin the preserved samples and all were reduced to 10 cfu/g within oneweek. Although S. aureus and C. albicans died off within one week in theunpreserved sample, there was some differentiation between preserved andunpreserved samples at 24 hours.

TABLE 18 Inoculum - Colony Forming Units Added per Gram (CFU/g) ofMake-up Remover, pH 8.1 Challenge #1 Challenge #2 Organism CFU/g CFU/gS. aureus 2.4 × 10⁶ 4.4 × 10⁶ K. pneumoniae + 4.0 × 10⁶ 4.0 × 10⁶ E.gergoviae P. aeruginosa + 3.8 × 10⁶ 5.1 × 10⁶ B. cepacia C. albicans 1.3× 10⁵ 4.2 × 10⁵ Mixed Mold 2.3 × 10⁵ 4.3 × 10⁵

TABLE 19 Inoculum Recovered from Make-up Remover, pH 8.1 at ‘0’ Hour -Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge #2 OrganismCFU/g CFU/g S. aureus 7.3 × 10⁵ 9.4 × 10⁵ K. pneumoniae + 9.4 × 10⁵ 8.8× 10⁶ E. gergoviae P. aeruginosa + 7.0 × 10⁵ 6.2 × 10⁶ B. cepacia C.albicans 3.4 × 10⁴ 2.2 × 10⁵ Mixed Mold 6.3 × 10⁴ 1.3 × 10⁵

TABLE 20a Unpreserved Make-up Remover, pH 8.1 - Colony Forming Units perGram (CFU/g) Challenge #1 24 48 2 3 4 Organism Hours Hours 1 Week WeeksWeeks Weeks S. aureus 1.0 × 10² — <10 <10 <10 <10 K. pneumoniae + 5.1 ×10⁶ — 8.0 × 10⁶ 6.8 × 10⁶ 7.0 × 10⁶ 2.5 × 10⁶ E. gergoviae P.aeruginosa + 4.5 × 10⁶ — 6.6 × 10⁶ 1.5 × 10⁶ 1.6 × 10⁶ 1.5 × 10⁶ B.cepacia C. albicans 4.0 × 10² — <10 <10 <10 <10 Mixed Mold 1.1 × 10⁴ 3.7× 10⁴ 2.5 × 10⁴ 2.9 × 10⁴ 7.0 × 10⁴ 2.0 × 10⁴ Challenge #2 24 48 2 3 4Organism Hours Hours 1 Week Weeks Weeks Weeks S. aureus <10 — <10 <10<10 <10 K. pneumoniae + 5.5 × 10⁶ — 9.2 × 10⁶ 1.7 × 10⁶ 2.2 × 10⁶ 8.0 ×10⁵ E. gergoviae P. aeruginosa + 9.8 × 10⁶ — 8.7 × 10⁶ 2.0 × 10⁶ 6.8 ×10⁶ 3.2 × 10⁶ B. cepacia C. albicans 9.1 × 10³ — 1.4 × 10³ 2.5 × 10² <10<10 Mixed Mold 2.7 × 10⁵ 1.0 × 10⁵ 1.5 × 10⁵ 1.0 × 10⁵ 1.0 × 10⁵ 1.0 ×10⁵

TABLE 20b Make-up Remover, pH 8.1 with 1% ECT-4 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4Organism Hours Hours Week Weeks Weeks Weeks Hours Hours Weeks WeeksWeeks Weeks S. aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K.pneumoniae + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae P.aeruginosa + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia C.albicans <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold <10 <10<10 <10 <10 <10 <10 <10 <10 <10 <10 <10

TABLE 20c Make-up Remover, pH 8.1 with 1% ECT-6 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4Organism Hours Hours Week Weeks Weeks Weeks Hours Hours Week Weeks WeeksWeeks S. aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 P.aeruginosa + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia K.pneumoniae + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae C.albicans <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold 7.0 ×10¹ <10 <10 <10 <10 <10 2.6 × 10³ <10 <10 <10 <10 <10

Conclusions: The ECT preservative blends can effectively protectcosmetic formulations against bacterial and fungal growth, at moderateuse levels even in alkaline pH.

Example 3 Microbiological Efficacy—0.6% Preservation Blends

To ascertain an effective use range, Example 2 was repeated with blendECT 4 at 0.6% dose rate.

Summary of Test Results:

ECT 4 tested at 0.6%, was effective in 4 of the 5 personal careformulations meeting CTFA recommendations for at least a 99.9% reductionof vegetative bacteria and at least a 90% reduction of yeasts and moldswithin 7 days following each challenge with no increase in countthereafter. It was not however, effective in the high pH Make-up Removerformulation.

Hair Conditioner, pH 3.99, AR13-069 (Same as AR5-024) (Data Shown inTables 21 Through 23b)

Excellent reduction of all challenge organisms occurred within 24 hoursin the preserved samples. All challenge organisms were reduced to <10cfu/g within one week following each challenge. Formulation met CTFArecommendations.

TABLE 21 Inoculum - Colony Forming Units Added per Gram (CFU/g) of HairConditioner Challenge #1 Challenge #2 Organism CFU/g CFU/g S. aureus 1.6× 10⁶ 1.1 × 10⁶ K. pneumoniae + 1.6 × 10⁶ 1.9 × 10⁶ E. gergoviae P.aeruginosa + 3.4 × 10⁶ 1.8 × 10⁶ B. cepacia C. albicans 1.5 × 10⁵ 8.2 ×10⁴ Mixed Mold 7.2 × 10⁴ 5.7 × 10⁴

TABLE 22 Inoculum Recovered from Unpreserved Hair Conditioner at ‘0’Hour - Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge #2Organism CFU/g CFU/g S. aureus 1.9 × 10⁶ 4.0 × 10⁷ K. pneumoniae + 1.2 ×10⁶ 1.0 × 10⁸ E. gergoviae P. aeruginosa + 2.2 × 10⁶ 1.5 × 10⁸ B.cepacia C. albicans 2.4 × 10⁵ 1.3 × 10⁸ Mixed Mold 1.0 × 10⁵ 8.1 × 10⁴

TABLE 23a Unpreserved Hair Conditioner - Colony Forming Units per Gram(CFU/g) Challenge #1 24 48 1 2 3 4 Organism Hours Hours Week Weeks WeeksWeeks S. aureus 1.7 × 10⁴ — 8.0 × 10⁶ >10⁷ 4.6 × 10⁷ 5.6 × 10⁷ K.pneumoniae + 3.7 × 10⁶ — >3.0 × 10⁷   1.8 × 10⁸ 8.6 × 10⁷ 5.6 × 10⁷ E.gergoviae P. aeruginosa + 2.6 × 10⁷ — 1.1 × 10⁸ 1.3 × 10⁸ 1.5 × 10⁸ 1.3× 10⁸ B. cepacia C. albicans 3.7 × 10⁵ — 8.8 × 10⁶  3.7 × 10⁶*  7.2 ×10⁶*  5.8 × 10⁶* (3.4 × 10⁶⁾ (5.4 × 10⁷⁾ (7.6 × 10⁷⁾ Mixed Mold 3.4 ×10⁴ 2.2 × 10⁴ 3.1 × 10⁴ 1.0 × 10⁴ 3.0 × 10³ 3.9 × 10³ Challenge #2 24 481 2 3 4 Organism Hours Hours Week Weeks Weeks Weeks S. aureus 3.6 × 10⁷— 7.1 × 10⁷ 4.9 × 10⁷ 4.9 × 10⁷ 3.5 × 10⁷ K. pneumoniae + 9.3 × 10⁷ —1.1 × 10⁸ 7.6 × 10⁷ 6.6 × 10⁷ 6.2 × 10⁷ E. gergoviae P. aeruginosa + 1.1× 10⁸ — 1.0 × 10⁸ 3.1 × 10⁸ 2.3 × 10⁸ 3.0 × 10⁸ B. cepacia C. albicans 8.8 × 10⁶* —  5.3 × 10⁶*  7.0 × 10⁶*  1.1 × 10⁶*  3.8 × 10⁶* (1.2 ×10⁸⁾ (9.0 × 10⁷⁾ (1.2 × 10⁸⁾ (>3.0 × 10⁶⁾   (>3.0 × 10⁷⁾   Mixed Mold3.7 × 10⁴ 2.8 × 10⁴ 2.5 × 10⁴ 1.2 × 10⁴ 1.4 × 10⁴ 1.1 × 10⁴

TABLE 23b Hair Conditioner with 0.6% ECT 4- Colony Forming Units perGram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4Organism Hours Hours Week Weeks Weeks Weeks Hours Hours Week Weeks WeeksWeeks S. aureus <10 — <10 — — <10 <10 — <10 <10 <10 <10 K. pneumoniae +<10 — <10 — — <10 <10 — <10 <10 <10 <10 E. gergoviae P. aeruginosa + <10— <10 — — <10 <10 — <10 <10 <10 <10 B. cepacia C. albicans <10 — <10 — —<10 <10 — <10 <10 <10 <10 Mixed Mold <10 <10 <10 — — <10 <10 <10 <10 <10<10 <10

Make-Up Remover, pH 5.15, AR12-067 (Ref # KKL9-181), (Data Shown inTables 24 Through 26b)

Excellent reduction of all challenge organisms occurred within 24 hourswith 0.6% ECT 4 blend. All challenge organisms were reduced to <10 cfu/gwithin one week following each challenge. Formulation met CTFArecommendations.

TABLE 24 Inoculum - Colony Forming Units Added per Gram (CFU/g) ofMake-up Remover Challenge #1 Challenge #2 Organism CFU/g CFU/g S. aureus1.6 × 10⁶ 1.1 × 10⁶ K. pneumoniae + 1.6 × 10⁶ 1.9 × 10⁶ E. gergoviae P.aeruginosa + 3.4 × 10⁶ 1.8 × 10⁶ B. cepacia C. albicans 1.5 × 10⁵ 8.2 ×10⁴ Mixed Mold 7.2 × 10⁴ 5.7 × 10⁴

TABLE 25 Inoculum Recovered from Unpreserved Make-up Remover, pH 5.15 at‘0’ Hour Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge #2Organism CFU/g CFU/g S. aureus 3.7 × 10⁵ 6.9 × 10⁵ K. pneumoniae + 5.5 ×10⁵ 7.4 × 10⁵ E. gergoviae P. aeruginosa + 9.0 × 10⁵ 4.9 × 10⁶ B.cepacia C. albicans 2.0 × 10⁵ 1.1 × 10⁵ Mixed Mold 6.5 × 10⁴ 9.7 × 10⁴

TABLE 26a Unpreserved Make-up Remover at pH 5.15 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 24 48 1 2 3 4 Organism Hours Hours WeekWeeks Weeks Weeks S. aureus 2.3 × 10² — <10 <10 <10 <10 K. pneumoniae +2.6 × 10⁵ — 1.5 × 10⁴ <10 1.0 × 10¹ <10 E. gergoviae P. aeruginosa + 2.8× 10⁵ — 4.3 × 10⁶ 5.8 × 10⁶ 4.3 × 10⁶ 2.8 × 10⁶ B. cepacia C. albicans1.3 × 10⁵ — 4.3 × 10⁴ 5.5 × 10⁴ 3.8 × 10⁴ 4.2 × 10⁴ Mixed Mold 6.5 × 10⁴4.1 × 10⁴ 6.5 × 10⁴ 3.2 × 10⁴ 6.5 × 10⁴ 5.7 × 10⁴ Challenge #2 24 48 1 23 4 Organism Hours Hours Week Weeks Weeks Weeks S. aureus 1.7 × 10³ —<10 <10 <10 <10 K. pneumoniae + 1.8 × 10⁵ — 6.1 × 10³ 1.0 × 10¹ <10 <10E. gergoviae P. aeruginosa + 4.8 × 10⁶ — 6.0 × 10⁶ 6.3 × 10⁶ 5.2 × 10⁶7.1 × 10⁶ B. cepacia C. albicans 5.6 × 10⁴ —  1.6 × 10³*  2.3 × 10⁵* 5.0 × 10⁴*  5.2 × 10⁴* (>3.0 × 10⁵)   (>3.0 × 10⁵)   (4.6 × 10⁶) (4.6 ×10⁶) Mixed Mold 7.9 × 10⁴ 4.7 × 10⁴ 1.1 × 10⁵ 7.2 × 10⁴ 1.2 × 10⁵ 5.5 ×10⁴

TABLE 26b Make-up Remover with 0.6% ECT4, pH 5.15 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4Organism Hours Hours Week Weeks Weeks Weeks Hours Hours Week Weeks WeeksWeeks S. aureus <10 — <10 — — <10 <10 — <10 <10 <10 <10 K. pneumoniae +<10 — <10 — — <10 <10 — <10 <10 <10 <10 E. gergoviae P. aeruginosa + <10— <10 — — <10 <10 — <10 <10 <10 <10 B. cepacia C. albicans <10 — <10 — —<10 <10 — <10 <10 <10 <10 Mixed Mold 1.3 × 10² 1.0 × 10¹ <10 — — <10 <10<10 <10 <10 <10 <10

(i) Lotion, pH 7.85, KKL 14-46 (Data Shown in Tables 27 Through 29b)

This formulation was contaminated before the start of this testing andwas previously noted as being more susceptible to contamination than theother four products tested. Excellent reduction of all challengeorganisms by 0.6% ECT 4 occurred within 24 hours. All challengeorganisms were reduced to <10 cfu/g within one week following eachchallenge. Formulation met CTFA recommendations.

TABLE 27 Inoculum - Colony Forming Units Added per Gram (CFU/g) ofProduct Challenge #1 Challenge #2 Organism CFU/g CFU/g S. aureus 1.5 ×10⁶ 1.6 × 10⁶ K. pneumoniae + 1.7 × 10⁶ 1.6 × 10⁶ E. gergoviae P.aeruginosa + 2.3 × 10⁶ 2.6 × 10⁶ B. cepacia C. albicans 5.2 × 10⁴ 7.1 ×10⁴ Mixed Mold 1.9 × 10⁴ 3.9 × 10⁴

TABLE 28 Inoculum Recovered from Unpreserved Lotion at ‘0’ Hour - ColonyForming Units per Gram (CFU/g) Challenge #1 Challenge #2 Organism CFU/gCFU/g S. aureus >3.0 × 10⁷ 1.5 × 10⁷ K. pneumoniae + >3.0 × 10⁷ 2.1 ×10⁷ E. gergoviae P. aeruginosa +  2.9 × 10⁷ 1.5 × 10⁷ B. cepacia C.albicans  1.0 × 10⁶ (2.5 × 10⁷*) 4.2 × 10⁵ (5.8 × 10⁶*) Mixed Mold  2.3× 10⁴ (>3.0 × 10⁷*) 3.0 × 10⁴ (6.3 × 10⁶*) *bacterial contaminant

TABLE 29a Unpreserved Lotion - Colony Forming Units per Gram (CFU/g)Challenge #1 24 48 1 2 3 4 Organism Hours Hours Week Weeks Weeks WeeksS. aureus >3.0 × 10⁷ — 1.4 × 10⁷ 2.9 × 10⁷ 4.3 × 10⁷ 2.6 × 10⁷ K.pneumoniae + >3.0 × 10⁷ — 2.4 × 10⁷ 3.0 × 10⁷ 3.4 × 10⁷ 1.7 × 10⁷ E.gergoviae P. aeruginosa +  2.9 × 10⁷ — 3.2 × 10⁷ 2.3 × 10⁷ 2.0 × 10⁷ 3.0× 10⁷ B. cepacia C. albicans  1.2 × 10⁶* — 1.3 × 10⁷ 7.0 × 10⁵ 4.3 × 10⁵3.1 × 10⁵ (>3.0 × 10⁷) (9.9 × 10⁶) (6.9 × 10⁶) (>3.0 × 10⁶)   Mixed Mold 1.2 × 10⁴*  1.7 × 10⁴*  1.8 × 10⁴*  4.0 × 10⁴*  1.7 × 10⁴*  1.0 × 10⁴*(>3.0 × 10⁷) (2.0 × 10⁷) (>3.0 10⁵) (>3.0 10⁶) (>3.0 10⁶) (>3.0 10⁶)Challenge #2 24 48 1 2 3 4 Organism Hours Hours Week Weeks Weeks WeeksS. aureus 2.7 × 10⁷ — 2.1 × 10⁷ 3.3 × 10⁷ 2.4 × 10⁷ 2.7 × 10⁷ K.pneumoniae + 2.1 × 10⁷ — 1.4 × 10⁷ 2.7 × 10⁷ 2.2 × 10⁷ 1.4 × 10⁷ E.gergoviae P. aeruginosa + 2.0 × 10⁷ — 2.0 × 10⁷ 3.8 × 10⁷ 3.0 × 10⁷ 3.7× 10⁷ B. cepacia C. albicans 4.8 × 10⁵ — 5.0 × 10⁵ 2.6 × 10⁵ 2.2 × 10⁵2.9 × 10⁵ (>3.0 10⁶) (>3.0 10⁶) (>3.0 10⁶) (>3.0 10⁶) (>3.0 10⁶) MixedMold  3.0 × 10⁴* 3.1 × 10⁴*  2.4 × 10⁴*  1.1 × 10⁵*  3.1 × 10⁴*  1.1 ×10⁵* (>3.0 10⁶) (>3.0 10⁶) (>3.0 10⁶) (>3.0 10⁶) (>3.0 10⁶) (>3.0 10⁶) *Bacterial Contamination

TABLE 29b Lotion with 0.6% ECT 4- Colony Forming Units per Gram (CFU/g)Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4 Organism HoursHours Week Weeks Weeks Weeks Hours Hours Week Weeks Weeks Weeks S.aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K. pneumoniae + <10 —<10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae P. aeruginosa + <10 —<10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia C. albicans <10 — <10<10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold 2.0 × 10¹ <10 <10 <10 <10<10 1.4 × 10² <10 <10 <10 <10 <10

(i) Water in Oil Emulsion, pH N/A, AR12-068 (Data Shown in Tables 30Through 32b)

Although S. aureus, K. pneumoniae/E. gergoviae, and Ps. aeruginosa/B.cepacia inocula died off within one week in the unpreserved samplefollowing the first challenge, all the challenge organisms in thepreserved samples were reduced to <10 cfu/g within 48 hours. Formulationmet CTFA recommendations.

TABLE 30 Inoculum - Colony Forming Units Added per Gram (CFU/g) ofProduct Challenge #1 Challenge #2 Organism CFU/g CFU/g S. aureus 1.5 ×10⁶ 1.6 × 10⁶ K. pneumoniae + 1.7 × 10⁶ 1.6 × 10⁶ E. gergoviae P.aeruginosa + 2.3 × 10⁶ 2.6 × 10⁶ B. cepacia C. albicans 5.2 × 10⁴ 7.1 ×10⁴ Mixed Mold 1.9 × 10⁴ 3.9 × 10⁴

TABLE 31 Inoculum Recovered from Unpreserved Water in Oil Emulsion (68A)at ‘0’ Hour - Colony Forming Units per Gram (CFU/g) Challenge #1Challenge #2 Organism CFU/g CFU/g S. aureus 4.2 × 10⁵ 1.5 × 10⁵ K.pneumoniae + 5.5 × 10⁵ 2.4 × 10⁵ E. gergoviae P. aeruginosa + 1.2 × 10⁵1.2 × 10⁵ B. cepacia C. albicans 4.0 × 10⁴ 1.2 × 10⁴ Mixed Mold 1.7 ×10⁴ 3.9 × 10⁴

TABLE 32a Unpreserved Water in Oil Emulsion - Colony Forming Units perGram (CFU/g) Challenge #1 24 48 1 2 3 4 Organism Hours Hours Week WeeksWeeks Weeks S. aureus 7.0 × 10⁴ — <10 <10 <10 <10 K. pneumoniae + 1.0 ×10¹ — <10 <10 <10 <10 E. gergoviae P. aeruginosa + <10 — <10 <10 <10 <10B. cepacia C. albicans 8.0 × 10² — 1.8 × 10² 2.7 × 10² 2.1 × 10² 3.2 ×10² Mixed Mold 2.8 × 10³ 2.9 × 10³ 1.8 × 10⁴ 1.9 × 10⁴ 5.1 × 10² 1.1 ×10³ Challenge #2 24 48 1 2 3 4 Organism Hours Hours Week Weeks WeeksWeeks S. aureus 2.4 × 10⁵ — <10 <10 <10 <10 K. pneumoniae + 8.0 × 10¹ —<10 <10 <10 <10 E. gergoviae P. aeruginosa + 6.0 × 10¹ — <10 4.0 × 10¹<10 <10 B. cepacia C. albicans 1.0 × 10⁴ — 8.5 × 10³ 5.4 × 10³ 6.9 × 10³6.6 × 10³ Mixed Mold 2.2 × 10⁴ 2.7 × 10⁴ 2.0 × 10⁴ 2.3 × 10⁴ 2.8 × 10⁴1.9 × 10⁴

TABLE 32b Water in Oil Emulsion with 0.6% ECT 4 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4Organism Hours Hours Week Weeks Weeks Weeks Hours Hours Week Weeks WeeksWeeks S. aureus <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 K.pneumoniae + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 E. gergoviae P.aeruginosa + <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 B. cepacia C.albicans <10 — <10 <10 <10 <10 <10 — <10 <10 <10 <10 Mixed Mold 7.0 ×10¹ <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10

(i) Make-Up Remover, pH 8.1, KKL 14-45 (Data Shown in Tables 33 Through35b)

This formulation with 0.6% ECT 4 did not meet the CTFA recommendationsfor a 90% decrease in fungi within 1 week. The unpreserved samples hadvery little survival of the Staph, Klebsiella, Pseudomonas andEnterobacter; but the Pseudomonas, Burkholderia, yeast and fungi didsurvive. Formulation did not meet CTFA recommendations, whereas a 1%dose gave good activity (see Table 20b). Testing was discontinued beforethe second challenge was to be done due to failure of product.

TABLE 33 Inoculum - Colony Forming Units Added per Gram (CFU/g) ofMake-up Remover, pH 8.1 (45A-C) Challenge #1 Challenge #2 Organism CFU/gCFU/g S. aureus 1.5 × 10⁶ — K. pneumoniae + 1.7 × 10⁶ — E. gergoviae P.aeruginosa + 2.3 × 10⁶ — B. cepacia C. albicans 5.2 × 10⁴ — Mixed Mold1.9 × 10⁴ —

TABLE 34 Inoculum Recovered from Make-up Remover, pH 8.1 (45A) at ‘0’Hour - Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge #2Organism CFU/g CFU/g S. aureus 6.1 × 10³ — K. pneumoniae + 4.7 × 10⁵ —E. gergoviae P. aeruginosa + 5.2 × 10² — B. cepacia C. albicans 1.2 ×10⁴ — Mixed Mold 6.5 × 10⁴ —

TABLE 35a Unpreserved Make-up Remover, pH 8.1 - Colony Forming Units perGram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4Organism Hours Hours Week Weeks Weeks Weeks Hours Hours Week Weeks WeeksWeeks S. aureus <10 — <10 <10 <10 <10 Test terminated K. pneumoniae +4.0 × 10¹ — <10 <10 <10 <10 E. gergoviae P. aeruginosa + <10 — lawn lawn4.3 × 10⁶ 2.5 × 10⁶ B. cepacia C. albicans 8.1 × 10² — 2.6 × 10² 2.0 ×10¹ <10 <10 Mixed Mold 4.7 × 10⁴ 4.1 × 10⁴ 4.7 × 10⁴ 2.0 × 10⁴ 1.9 × 10⁴6.3 × 10³

TABLE 35b Make-up Remover, pH 8.1 with 0.6% ECT 4 - Colony Forming Unitsper Gram (CFU/g) Challenge #1 Challenge #2 24 48 1 2 3 4 24 48 1 2 3 4Organism Hours Hours Week Weeks Weeks Weeks Hours Hours Week Weeks WeeksWeeks S. aureus <10 — <10 <10 — <10 Test terminated K. pneumoniae + 3.0× 10¹ — <10 <10 — <10 E. gergoviae P. aeruginosa + <10 — <10 <10 — <10B. cepacia C. albicans 1.4 × 10³ — 4.0 × 10¹ <10 — <10 Mixed Mold 2.1 ×10⁴ 4.2 × 10⁴ 1.5 × 10⁴ 1.5 × 10⁴ — 4.0 × 10³

To determine if the unexpected activity of ECT 4 at high pH (8.1) can beattributed solely to the activity of benzyl alcohol the high pH make-upremover was preserved with 1% ECT4 and with 0.83% benzyl alcohol(equivalent amount found in 1% ECT4).

Summary of Results:

The data shows that ECT4 is more effective than its benzyl alcoholalone, Tables 39 and 40 show that benzyl alcohol alone was notsufficiently efficacious against the mixed mold challenge. High pHMake-up Remover preserved only with benzyl alcohol did not meet CTFArecommendations for a minimum of 90% mold reduction in 7 days; thesample with 1% ECT4 does meet this criterion. This is unexpected as theorganic acids in ECT4 would not be expected to have any activity at thishigh a pH.

TABLE 36 Inoculum - Colony Forming Units Added per Gram (CFU/g) ofProduct Organism Challenge #1 Challenge #2 S. aureus 2.5 × 10⁶ 2.0 × 10⁶P. aeruginosa + 3.7 × 10⁶ 3.0 × 10⁶ B. cepacia K. pneumoniae + 1.8 × 10⁶3.0 × 10⁶ E. gergoviae C. albicans 1.3 × 10⁵ 1.6 × 10⁵ Mixed Molds 1.1 ×10⁵ 2.2 × 10⁴

TABLE 37 Inoculum Recovered from unpreserved product at ‘0’ Hour -Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge #2 OrganismCFU/g CFU/g S. aureus 5.5 × 10⁴ NP P. aeruginosa + 2.3 × 10⁵ NP B.cepacia K. pneumoniae + 3.7 × 10⁵ NP E. gergoviae C. albicans 7.4 × 10⁵NP Mixed molds 9.0 × 10⁴ NP NP = Not Plated, contaminant overgrewchallenge bacteria

TABLE 38 Unpreserved - Colony Forming Units per Gram (CFU/g) Challenge #1 Challenge # 2 Test 24 48 7 14 21 28 24 48 7 14 21 28 Organism HoursHours Days Days Days Days Hours Hours Days Days Days Days S. aureus 3.7× 10⁴ — * * NP NP NP — NP NP NP NP P. aeruginosa + 6.7 × 10⁴ — * * NP NPNP — NP NP NP NP B. cepacia K. pneumoniae + 4.0 × 10⁵ — * * NP NP NP —NP NP NP NP E. gergoviae C. albicans 2.7 × 10⁵ — * * NP NP NP — NP NP NPNP Mixed Molds 8.0 × 10⁴ 2.0 × 10⁵ * * NP NP NP NP NP NP NP NP *Contaminant overgrew challenge bacteria NP = Not Plated, contaminantovergrew challenge bacteria

TABLE 39 Make-up Remover with 1% ECT 4- Colony Forming Units per Gram(CFU/g) Challenge # 1 Test 24 48 7 14 21 28 Organism Hours Hours DaysDays Days Days S. aureus <10 — <10 <10 <10 <10 P. aeruginosa + <10 — <10<10 <10 <10 B. cepacia K. pneumoniae + 2.1 × 10³ — <10 <10 <10 <10 E.gergoviae C. albicans 1.8 × 10⁵ — <10 <10 <10 <10 Mixed Molds 5.0 × 10⁴5.0 × 10⁴ 3.0 × 10³ 4.2 × 10² 2.8 × 10² 2.1 × 10² Challenge #2 Test 2448 7 14 21 28 Organism Hours Hours Days Days Days Days S. aureus NP <10<10 <10 <10 <10 P. aeruginosa + NP <10 <10 <10 <10 <10 B. cepacia K.pneumoniae + NP <10 <10 <10 <10 <10 E. gergoviae C. albicans NP <1.0 ×10²   <10 <10 <10 <10 Mixed Molds NP 1.0 × 10² 3.0 × 10¹ 6.0 × 10¹ 1.0 ×10¹ <10 NP = Not plated

TABLE 40 Make-up Remover at pH 8.1 with 0.83% Benzyl Alcohol - ColonyForming Units per Gram (CFU/g) Challenge # 1 Test 24 48 7 14 21 28Organism Hours Hours Days Days Days Days S. aureus <10 — <10 <10 <10 <10P. aeruginosa + <10 — <10 <10 <10 <10 B. cepacia K. pneumoniae + <10 —<10 <10 <10 <10 E. gergoviae C. albicans 3.1 × 10⁴ — 1.6 × 10² <10 <10<10 Mixed Molds 2.1 × 10⁵ 1.2 × 10⁵ 3.0 × 10⁴ 8.0 × 10⁴ 1.5 × 10⁴ 5.1 ×10³ Challenge # 2 Test 24 48 7 14 21 28 Organism Hours Hours Days DaysDays Days S. aureus NP <10 <10 <10 <10 <10 P. aeruginosa + NP <10 <10<10 <10 <10 B. cepacia K. pneumoniae + NP <10 <10 <10 <10 <10 E.gergoviae C. albicans NP <1.0 × 10²   <10 <10 <10 <10 Mixed Molds NP 3.8× 10³ 3.4 × 10³ 3.6 × 10³ 9.0 × 10³ 4.0 × 10² NP = Not plated

While the invention has been described above with reference to specificembodiments thereof, it is apparent that many changes, modifications,and variations can be made without departing from the inventive conceptdisclosed herein. Accordingly, it is intended to embrace all suchchanges, modifications and variations that fall within the spirit andbroad scope of the appended claims. All patent applications, patents andother publications cited herein are incorporated by reference in theirentirety.

What is claimed is:
 1. A composition having effective broad spectrumpreservation activity comprising: (a) benzyl alcohol, (b) salicylicacid, (c) sorbic acid and (d) a compound selected from the groupconsisting of 1,3-propanediol, glycerin and a combination thereof,wherein component (a) is present at a concentration of from about 70% toabout 90% by weight, component (b) is present at a concentration of fromabout 1% to 15% by weight, component (c) is present at a concentrationof from about 1% to 4% by weight, and component (d) is present at aconcentration of from about 1% to 15% by weight, provided that the totalamount of components (b) and (c) is no more than 15% by weight, allbased on the total weight of the composition.
 2. The composition ofclaim 1, further comprising water in an amount less than 1000 parts permillion.
 3. The composition of claim 1, wherein the total weightpercentage of component (a), (b), (c) and (d) combined is 100%.
 4. Thecomposition of claim 1, wherein component (a) is present at aconcentration of from about 77% to 86% by weight, component (b) ispresent at a concentration of from about 3% to about 12% by weight,component (c) is present at a concentration of from about 2.5% to 3.5%by weight, and component (d) is present at a concentration of from about1% to 10% by weight, based on the total weight of the composition. 5.The composition of claim 1, wherein component (d) is glycerin present ata concentration of from about 2% to about 5%.
 6. The composition ofclaim 1 wherein component (d) is 1,3-propanediol presented at aconcentration of from about 2% to about 5%.
 7. The composition of claim1, further comprising dehydroacetic acid, wherein the total amount ofsalicylic acid, sorbic acid, and dehydroacetic acid is no more than 15wt % of the composition.
 8. The composition of claim 7, wherein thedehydroacetic acid and the component (d) are present at a weight ratiorange of from about 3:1 to 1:1.
 9. The composition of claim 1, whereincomponent (a) is present at a concentration of from about 77% to about86%, component (b) is present at a concentration of from about 8% to11%, component (c) is present at concentration of from about 2.5% to3.5%, and component (d) is glycerin present at a concentration of fromabout 2% to about 5%, all based on the weight of the composition.
 10. Atopical formulation comprising the preservative composition of claim 1and additives selected from the group consisting of solvents,surfactants, emulsifiers, consistency factors, conditioners, emollients,skin caring ingredients, moisturizers, thickeners, lubricants, fillers,anti-oxidants, other preservatives, active ingredients, fragrances andmixtures thereof.
 11. The topical formulation of claim 10, wherein thepreservative composition is present at a concentration of from about0.5% to about 1.5% based on the weight of the formulation.
 12. Thetopical formulation of claim 11, wherein the preservative composition ispresent at a concentration of from about 0.6% to about 1% based on theweight of the formulation.
 13. The topical formulation of claim 10,wherein the formulation is in the form of an oil-in-water emulsion. 14.The topical formulation of claim 10 wherein the formulation is in theform of a water-in-oil emulsion.
 15. A composition having effectivebroad spectrum preservation activity consisting of: (a) benzyl alcohol,(b) salicylic acid, (c) sorbic acid and (d) a compound selected from thegroup consisting of 1,3-propanediol, glycerin and a combination thereof;and (e) optionally, dehydroacetic acid, wherein component (a) is presentat a concentration of from about 70% to about 90% by weight, component(b) is present at a concentration of from about 1% to 15% by weight,component (c) is present at a concentration of from about 1% to 4% byweight, and component (d) is present at a concentration of from about 1%to 15% by weight, and wherein total amount of components (b), (c) and(a) is no more than 15% by weight, all based on the total weight of thecomposition.
 16. The composition of claim 15, wherein component (a) ispresent at a concentration of from about 77% to 86% by weight, component(b) is present at a concentration of from about 3% to about 12% byweight, component (c) is present at a concentration of from about 2.5%to 3.5% by weight, and component (d) is present at a concentration offrom about 1% to 10% by weight, based on the total weight of thecomposition.
 17. The composition of claim 15, further comprisingdehydroacetic acid, wherein the total amount of salicylic acid, sorbicacid, and dehydroacetic acid is no more than 15 wt % of the composition.18. A topical formulation comprising the preservative composition ofclaim 15 and additives selected from the group consisting of solvents,surfactants, emulsifiers, consistency factors, conditioners, emollients,skin caring ingredients, moisturizers, thickeners, lubricants, fillers,anti-oxidants, other preservatives, active ingredients, fragrances andmixtures thereof.
 19. The topical formulation of claim 18, wherein thepreservative composition is present at a concentration of from about0.5% to about 1.5% based on the weight of the formulation.
 20. Acomposition having effective broad spectrum preservation activitycomprising: (a) benzyl alcohol, (b) salicylic acid, (c) sorbic acid, (d)a compound selected from the group consisting of 1,3-propanediol,glycerin and a combination thereof, and (e) dehydroacetic acid, whereincomponent (a) is present at a concentration of from about 70% to about90% by weight, component (b) is present at a concentration of from about1% to 15% by weight, component (c) is present at a concentration of fromabout 1% to 4% by weight, and component (d) is present at aconcentration of from about 1% to 15% by weight, provided that the totalamount of components (b), (c), and (e) is no more than 15% by weight,all based on the total weight of the composition, and wherein water ispresent in an amount of less than 1000 parts per million.
 21. Thecomposition of claim 1, wherein the composition has a pH of greater than7.
 22. The composition of claim 15, wherein the composition has a pH ofgreater than 7.